Project Summary/abstract: There is no organ in the body that more heavily depends on a continuous supply of blood than the brain. The importance of the circulatory system to the brain is demonstrated by the fact that the brain makes up 2% of total body mass but it receives 20% of cardiac output. Furthermore, the brain does not contain local fuel reserves, as peripheral organs do. During behavior, brain regions are recruited for specific tasks and must be brought ?online? quickly. The brain regulates its own blood supply via a process called neurovascular coupling, in which neural activity rapidly increases local blood flow to meet moment-to-moment changes in regional brain energy demand. Impairments in neurovascular coupling contributes to neurodegeneration and vascular dementia. Neurovascular coupling is also the basis for functional brain imaging, which is currently the only way to infer brain-wide neuronal activation in humans. Despite the importance of understanding how the brain regulates its own blood supply, the molecular and cellular mechanisms underlying neurovascular coupling are still not clear. In this proposal, we have developed a two-photon in vivo imaging paradigm that can simultaneously measure neural activity (via a genetically encoded calcium indicator) and vascular dynamics (vessel diameter and blood flow) at single-vessel resolution in awake mice, enabling us to capture the neurovascular response to a natural stimulus (whisker stimulation) at high temporal and spatial resolution in vivo. We will combine this state-of-art in vivo imaging techniques with powerful molecular, electrophysiological, ultrastructural, and genetic approaches to elucidate the fundamental cellular mechanisms governing neurovascular coupling. Our preliminary data with in vivo live imaging and mouse genetics in the barrel cortex, suggest that endothelial cells (ECs) in the brain play active roles in neurovascular coupling. Traditionally, ECs were considered to be passively involved in neurovascular coupling, and to be a homogenous population. We found aECs and cECs display molecular and subcellular differences, and play distinct roles during neurovascular coupling. Our proposed work will identify the cellular, subcellular, and molecular mechanisms by which endothelial cells from different segments of the vascular tree initiate, transmit, and implement neurovascular coupling. Specifically, we will demonstrate how different type of ECs along the vascular tree mediate neurovascular coupling, thus connecting the dots between neural activity and local blood flow change. We expect that our ability to image CNS small blood vessels non-invasively in awake mice with high spatial and temporal resolution, along with other sophisticated techniques, will allow us to identify critical molecular and subcellular mechanisms that underlie neurovascular coupling in vivo. Moreover, our structural and molecular findings will provide tools that can be broadly used by the field to cerebrovascular biology. Finally, the data will provide critical information to accelerate our understanding and treatment of CNS diseases related to small vessels and vascular dementia.